Gas vesicles are buoyant intracellular organelles found in many aquatic bacteria including halobacteria, cyanobacteria and methanogens. Gas vesicles have the form of central cylinders with conical end caps and are synthesized bidirectionally from the apices of the cones, with elongation proceeding by growth of the cylindrical region. The vesicle membrane is impermeable to water, and accumulation of gases in the vesicle interior is thought to be a consequence of passive diffusion of gases across the membrane and exclusion of water during the synthetic process. Accumulation of gas vesicles increases cell buoyancy and facilitates movement of cells to the surface of liquid cultures. For Halobacterium halobium, an aerobe with a photophosphorylation system utilizing the light-driven proton pump, bacteriorhodopsin, the consequence of gas vesicle accumulation is the increased availability of oxygen for respiration and light for photophosphorylation.
H. halobium contains two varieties of gas vesicles, a major type that is shorter and wider and a minor form that is longer, more slender, and observed in small amounts in cultures of gas vesicle defective mutants. H. halobium displays extreme genetic instability for the gas vesicle phenotype (Vac) apparent upon visual inspection of colonies on agar plates. Inflated gas vesicles diffract light, giving an opaque appearance to Vac.sup.+ colonies, whereas Vac.sup.- mutants form translucent colonies. Spontaneous Vac.sup.- mutants are observed at a frequency of about 1%, and analysis of mutants has demonstrated that gas vesicle formation is determined or controlled by plasmid genes. Horne et al. (1988) Mol. Gen. Genet. 213, 459, have demonstrated that a plasmid gene encodes an 8.7 kD protein (GvpA, a constituent of the gas vesicle), and a chromosomal gene encodes a homologous 9 kD protein. The smaller plasmid encoded protein is constitutively produced in high amount while the chromosome encoded protein is produced only in the stationary phase, and probably accounts for the morphologically distinct gas vesicles observed in Vac.sup.- mutants.
Cloning of the major gas vesicle protein (GvpA)-encoding gene, gvpA, and analysis of Vac.sup.- mutants of H. halobium has resulted in the identification of a region of a 200-kb plasmid, pNRC100, which is important for gas vesicle synthesis. The nucleotide sequence of an 8520-bp region of pNRC100 has been reported which, including gvpA, contains a cluster of twelve genes organized into two divergent transcription units (Jones et al. [1991] Gene 102, 117, "Jones et al."). Analysis of spontaneously occurring mutants that are caused by the integration of insertion elements has suggested that several genes are involved in gas vesicle synthesis, but the role of the putative gene products in H. halobium is unclear.
Functional tests of cells transformed with vectors containing genomic DNA fragments of H. halobium have failed to identify the genetic information sufficient to direct gas vesicle formation. Blaseio et al. (1990) Proc Natl. Acad. Sci. 87, 6772 ("Blaseio et al."), have analyzed DNA fragments containing p-vac, a gene present on a 150-kb plasmid (pHH1) endogenous to H. halobium. The p-vac gene reportedly encodes a major structural protein of gas vesicles in H. halobium. H. halobium P03, a strain which lacks the p-vac region but contains a chromosomal c-vac, was transformed with a plasmid containing a 4.5-kb region of PHH1. The 4.5-kb region contained the entire p-vac region and 4-kb of flanking region. Transformants contained p-vac mRNA, but gas vesicles were not synthesized, indicating that expression of p-vac is not sufficient for gas vesicle formation. Blaseio et al. further report that transformation of Haloferax volcanii with a vector having an 11-kb fragment containing the mc-vac chromosomal gene from Haloferax mediterranei resulted in transformation to Vac.sup.+ phenotype as measured by the presence of light refractile bodies under phase-contrast microscopy. There is no evidence that gas vesicles were synthesized, or that the transformed cells were capable of flotation. The phase bright-inclusions observed by phase-contrast microscopy are not evidence of gas vesicles, and could result from production of polyhydroxyalkanoates (Garcia-Lillo et al. [1990] Applied and Environmental Microbiology 56, 2517). Horne et al. (1991) Mol. Microbiol. 5, 1159 ("Horne et al.") report the transformation of Haloferax volcanii with the vector of Blaseio et al. modified by deletions in the mc-vac fragment. None of the transformed cells synthesized gas vesicles. Horne et al. further report that transformation of Hf. volcanii with a construct containing a 6.8-kb fragment of the H. halobium plasmid pHH1 containing the p-vac gene and significant amounts of flanking sequence failed to enable gas vesicle synthesis.
In accordance with the present invention, all of the genetic information necessary to direct the synthesis of gas vesicles has been identified. Furthermore, a method is provided for directing synthesis of gas vesicles in non-floating cells, and for transforming non-floating organisms to a floating phenotype.